Expansion microscopy enables high resolution imaging using conventional optical microscopes. The method was originally described by Chen et al 2015 (doi 10.1126/science.1260088). The main concept is to physically expand isotropically a biological sample several folds from the original size using a swellable polymer.
The increased resolution is achieved by physical moving the fluorophores apart through expansion of the sample, while maintaining the relative positions of the fluorophores. Therefore, the increase in resolution is directly proportional to the expansion factor.
Conventional protocol for ExM can be separated into 5 phases:
1) Immunofluorescence: affinity probes revealing the protein(s) of interest with fluorophores compatible with ExM.
2) Gelation: embed the sample in a swellable gel matrix
3) Anchoring: linking covalently the fluorophores present on the affinity probes to the gel matrix
4) Digestion: Thorough enzymatic digestion of proteins is performed to allow the gel to expand without constrains.
5) Expansion: allow the gel matrix to expand isotropically in 3D, retaining the fluorophores anchored to the gel polymer.
The retention of the fluorophores to the gel polymer is expected to happen in a fortuitous manner. This unsystematic strategy results in the loss of fluorophores. This is mainly occurring because the undirected chemistry used to couple fluorophores on conventional antibodies combined with the digestion step, leaves fluorescent protein peptides not anchored to the gel, and thus washed away.
We have developed a short cassette (termed “anchor” in the image above) that promotes the fluorescent dye to be retained on the swell polymer after protease digestion. We have equipped all of our secondary single-domain antibodies with this cassette to achieve a significant increase in performance compared to single-domain antibodies without this technology.
An additional feature of this technology is the ensure of the retention on samples that undergo post-fixation and an increment in the overall solubility allowing more efficient washing steps.